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Pe anti human cd3

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PE anti-human CD3 is a fluorescently labeled antibody that specifically binds to the CD3 antigen expressed on human T cells. It is a useful tool for the identification and enumeration of T cells in flow cytometry applications.

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Lab products found in correlation

Apc anti human cd19, by BioLegend (2 mentions) Fitc anti human cd45, by BioLegend (2 mentions) Apc anti human cd8, by BioLegend (2 mentions) Anti human cd45 apc h7, by BD (1 mentions) Facsaria 2, by BD (1 mentions) Allprep dna rna mini kit, by Qiagen (1 mentions) Anti human cd4 fitc, by Thermo Fisher Scientific (1 mentions) Gallios flow cytometer, by Beckman Coulter (1 mentions) Anti cd8 percp cy5, by BioLegend (1 mentions) Cytofix cytoperm fixation permeabilization solution, by Thermo Fisher Scientific (1 mentions) Anti cd4 apc, by BioLegend (1 mentions) Lsrfortessa flow cytometer, by BD (1 mentions) Lsrfortessatm cell analyzer, by BD (1 mentions) Anti cd4 apc, by Thermo Fisher Scientific (1 mentions) Anti cd69 fitc, by Miltenyi Biotec (1 mentions) Lsrfortessa, by BD (1 mentions) Fitc anti human cd4, by BioLegend (1 mentions) Pe anti human ifn γ, by BD (1 mentions) Pe anti human pd 1, by BioLegend (1 mentions) Pe cy7 anti human cd8, by BioLegend (1 mentions)

9 protocols using pe anti human cd3

1

Cell Isolation and Molecular Analysis

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Cells were isolated by the Ficoll-Paque method from peripheral blood (PB) and bone marrow (BM) aspirates. Cells were labeled with anti-human CD45 APC-H7 (BD Biosciences, clone 2D1, cat. 560178), anti-human CD3 PE (BioLegend, clone SK7, cat. 344806) and anti-human CD34 APC (eBioscience, Clone 4H11, cat. 17-0349-42). Cells were washed and resuspended in 4′,6-diamidino-2-phenylindole (DAPI) to exclude dead cells. Populations were sorted using a BD FACSAria-II (BD Biosciences). Total RNA and genomic DNA were extracted from bulk samples and sorted cells with AllPrep DNA/RNA Mini Kit (Qiagen, cat. 80204).
Sugita M., Wilkes D.C., Bareja R., Eng K.W., Nataraj S., Jimenez-Flores R.A., Yan L., De Leon J.P., Croyle J.A., Kaner J., Merugu S., Sharma S., MacDonald T.Y., Noorzad Z., Panchal P., Pancirer D., Cheng S., Xiang J.Z., Olson L., Van Besien K., Rickman D.S., Mathew S., Tam W., Rubin M.A., Beltran H., Sboner A., Hassane D.C., Chiosis G., Elemento O., Roboz G.J., Mosquera J.M, & Guzman M.L. (2021). Targeting the epichaperome as an effective precision medicine approach in a novel PML-SYK fusion acute myeloid leukemia. NPJ Precision Oncology, 5, 44.
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2

PBMC Immune Subset Characterization

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PBMC were collected from the rOoC and static plates and centrifuged at 300 g for 5 minutes. Next, cells were washed once with FACS buffer (PBS, supplemented with 1% BSA and 0.05% sodium azide) and centrifuged again at 300g for 5 min. Staining for immune subsets and activation markers was performed for 30 min, followed by a wash step with FACS buffer and analysis at a "Gallios" flow cytometer (Beckman Coulter Life Sciences, United States). The following antibodies were used at a dilution of 1 : 250 in FACS buffer:
• Anti-Human CD4 FITC (eBioscience 11-0049-42), Clone RPA-T4
• Anti-Human CD3 PE (BioLegend 300308), Clone HIT3a
• Anti-Human CD69 PE/Cy7 (Biolegend 310912), Clone FN50
• Anti-Human HLA-DR APC/Cy7 (Miltony Biotech 130-111-792), Clone REA805
• 7AAD ready-made solution (Sigma SML1633), dilution: 1 : 100
Busek M., Aizenshtadt A., Koch T., Frank A., Delon L., Martinez M.A., Golovin A., Dumas C., Stokowiec J., Gruenzner S., Melum E, & Krauss S. (2023). Pump-less, recirculating organ-on-a-chip (rOoC) platform. Lab on a chip, 23(4).
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3

Multiparametric Flow Cytometry Assay

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After culture, purified T cells were washed twice in PBS with 1% FBS and subsequently stained for 30 min at 4°C with the following fluorescein-conjugated monoclonal antibodies: human anti-CD3-PE (Biolegend, San Diego, CA), anti-CD4-APC (Biolegend, San Diego, CA), anti-CD8-PerCP/CY5.5(Biolegend and San Diego, CA). Stained cells were resuspended for 30 min at 4°C in Cytofix/Cytoperm fixation/permeabilization solution (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer's instructions. Once permeabilized, cells were washed twice and stained for intracellular cytokines with the following mAbs: human anti-IFNγ-FITC (Biolegend, San Diego, CA), anti–TNF-α-650TM (Biolegend, San Diego, CA). A total of 200,000 events were acquired by the BD LSRFortessa™ flow cytometer (BD Bioscience, San Jose, CA, USA). FlowJo v10.1 software (Tree Star, Ashland, OR, USA) was used for date analysis.
Xiang F., Cao X., Shen B., Chen X., Guo M., Ding X, & Zou J. (2020). Transcriptome Profiling Reveals Indoxyl Sulfate Should Be Culpable of Impaired T Cell Function in Chronic Kidney Disease. Frontiers in Medicine, 7, 178.
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4

Flow Cytometric Immune Cell Phenotyping

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Cells in staining buffer were obtained and stained for 30 min at 4 °C with the following fluorescein-conjugated monoclonal antibodies: human anti-CD3-PE (Biolegend, San Diego, CA), anti-CD4-APC (eBioscience, San Diego, CA), and anti-CD69-FITC (Miltenyi Biotech, Bergisch Gladbach, Germany). Then, the cells were analyzed on BD LSR FortessaTM cell analyzer (BD Bioscience). The data analysis was carried out with Flowjo v10.1 Software (Tree Star, Ashland, OR).
Chen R., Xiang F., Hu J., Cao X., Tan X., Jia P., Zhang T., Song N., Fang Y., Ding X, & Zou J. (2017). Factors associated with the elevated percentage of CD4CD69 T cells in maintained hemodialysis patients. Renal Failure, 39(1), 547-554.
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5

Quantitative Analysis of Blood Cell Types

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The L-PRF and H-PRF layers were obtained by centrifugation in 10-mL plastic tubes and divided into five equal portions. Cells from each layer were then centrifuged. Afterward, the cells were resuspended and incubated for 30 min on ice with FITC anti-human CD45 (1:200, Biolegend, no. 304006), PE anti-human CD3 (1:200, Biolegend, no. 300308), Pacific Blue™ anti-mouse/human CD11b (1:200, Biolegend, no. 101224), Alexa Fluor® 700 anti-human CD14 (1:200, Biolegend, no. 325614), PE/Cy7 anti-human CD16 (1:200, Biolegend, no. 302015) and APC anti-human CD19 (1:200, Biolegend, no.302212) antibodies to quantify the various blood cell types. Finally, flow-cytometric analysis was performed using a BD LSRFortessa instrument (USA), and the results were analyzed by FlowJo 10.
Feng M., Wang Y., Zhang P., Zhao Q., Yu S., Shen K., Miron R.J, & Zhang Y. (2020). Antibacterial effects of platelet-rich fibrin produced by horizontal centrifugation. International Journal of Oral Science, 12, 32.
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6

Comprehensive T Cell Phenotyping and Functional Analysis

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The following cell surface fluorochrome-conjugated monoclonal antibodies were used to detect T cells phenotype: FITC anti-human CD4, PE/Cy7 anti-human CD8, PE anti-human CD3 (Biolegend, San Diego, CA, USA) and APC anti-human NKG2D (eBioscience). T cell activity was determined by staining the surface APC anti-human CD69, intracellular PE anti-human IFN-γ (BD Biosciences, San Diego, CA, USA), PE anti-human GzmB (eBioscience, San Diego, CA, USA) and PE anti-human Bcl-2 (Biolegend) antibodies. PE anti-human PD-1 and APC anti-human Tim-3 were purchased from Biolegend. FITC anti-human CD45RA, Percp-cy5.5 anti-human CCR7 and PE anti-human CD45RO were purchased from Biolegend to determine T cell subsets. APC Annexin-V and 7-Aminoactinomycin D (7-ADD) from BD Biosciences were used for apoptosis staining. APC mouse IgG1, κ isotype, PE mouse IgG1 isotype, PE/Cy7 mouse IgG2a, κ isotype, FITC mouse IgG1 isotype, PE/Cy7 mouse IgG1, κ isotype (Biolegend) were used as controls. Flow cytometry was performed on the BD LSRFortessa flow cytometer, and data were analyzed using FlowJo Version 10 software.
He C., Zhou Y., Li Z., Farooq M.A., Ajmal I., Zhang H., Zhang L., Tao L., Yao J., Du B., Liu M, & Jiang W. (2020). Co-Expression of IL-7 Improves NKG2D-Based CAR T Cell Therapy on Prostate Cancer by Enhancing the Expansion and Inhibiting the Apoptosis and Exhaustion. Cancers, 12(7), 1969.
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7

Multicolor Flow Cytometry of Hematopoietic Cells

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The blood cells collected in heparin anticoagulant tubes were washed with PBS and incubated with the antibody panel for 30–45 minutes at 4°C in the dark. Then, the cells were lysed with Pharm Lyse (Cat# 555899, BD Biosciences, San Jose, CA, USA) for 4 minutes. After centrifugation, the samples were suspended in PBS for data acquisition by flow cytometry (BD FACSCanto II, Beckman Coulter). Fluorochrome-conjugated monoclonal antibodies to the following human or mouse antigens were used: FITC-anti mouse CD45 (Biolegend, San Diego, CA, USA, Cat# 147710, RRID: AB_2563541), PE-anti human CD45 (Biolegend, Cat# 304039, RRID: AB_314395), FITC-anti human CD45 (Biolegend, Cat# 304038, RRID: AB_314393), PE-anti human CD3 (Biolegend, Cat# 300308, RRID: AB_314043), APC-anti human CD19 (Biolegend, Cat# 392506, RRID:AB_2750096), PE-Cy7 anti human CD4 (Biolegend, Cat# 357410, RRID: AB_2565661), APC-anti human CD8 (Biolegend, Cat# 344722, RRID: AB_2075390). A FACSCalibur instrument and Cellquest software were used for flow cytometry analysis.
Li R., Qiu S., Yang W., Rao Z., Chen J., Yang Y., Zhu Q., Liu X., Bai Y, & Quan D. (2023). A comparative study of human and porcine-derived decellularised nerve matrices. Biomaterials Translational, 4(3), 180-195.
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8

Adenoma Characterization in Apc^(Min/+) Mice

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Conscious female ApcMin/+ mice (18-week old; n=2) received intravenous injections of FSNs (30 fmol g−1) via the tail vein using a tail-restrainer. The next day, the animals were deeply anesthetized using inhalant isofluorane and then euthanized by cervical dislocation. Small intestinal tissue containing adenomas was collected and sectioned into small (2–3 mm) pieces and then placed in a dounce glass homogenizer to create a cell suspension. Cells were passed through a 40-μm filter with Hank’s balanced salt solution (HBSS) containing DNAse. Cells were counted and resuspended in PBS at a concentration of 1 × 106 cells, prior to live dead staining with fixable LD aqua (#L34957, ThermoFisher Scientific, Waltham, MA) for 15 min. Cells were then washed and resuspended in PBS containing 2% bovine serum albumin (BSA), prior to staining with the fluorophore conjugated antibody panel (Pacific Blue anti-human CD11c (1:40; 117321), R-phycoerythrin (PE)-Cy7 anti-human Ly-6G (1:40; 127618), Allophycocyanin (APC)-Cy7 anti-human CD11b (1:40; 101225), PE anti-human CD3 (1:40; 100206), Peridinin-Chlorophyll protein (PerCP)-Cy5.5 anti-human CD45 (1:40; 103132); Biolegend, San Diego, CA) for 45 min. Cells were then washed and resuspended in 200 μL of PBS and then analyzed on a BD LSRII flow cytometer. All samples were analyzed in triplicate.
Rogalla S., Flisikowski K., Gorpas D., Mayer A.T., Flisikowska T., Mandella M.J., Ma X., Casey K.M., Felt S.A., Saur D., Ntziachristos V., Schnieke A., Contag C.H., Gambhir S.S, & Harmsen S. (2019). Biodegradable fluorescent nanoparticles for endoscopic detection of colorectal carcinogenesis. Advanced functional materials, 29(51), 1904992.
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9

Tumor Immune Profiling by Flow Cytometry

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Tumour samples were cut into small pieces and digested in RPMI-1640 supplemented with collagenase at 1 mg/mL (#C1889 Sigma USA). Digestion lasted for 1 hour at room temperature and filtered through a 70 µm filter. Single-cell suspensions were surface stained in fluorescence activated cell sorter (FACS) buffer (phosphate buffer solution (PBS), supplemented with 1% fetal bovine serum (FBS)) with the following antibodies for flow cytometric analysis, including FITC Zombie Green (#423 111 Biolegend USA), PE Anti-human CD3 (#344 806 Biolegend USA), APC Anti-human CD8 (#344 721 Biolegend USA), Brilliant Violet 421 Anti-human CD39 (#328 214 Biolegend USA), PE-Cy7 Anti-human PD-1 (#561 272 BD USA). Flow data were analysed by FlowJo V.10.0.
Liu T., Tan J., Wu M., Fan W., Wei J., Zhu B., Guo J., Wang S., Zhou P., Zhang H., Shi L, & Li J. (2020). High-affinity neoantigens correlate with better prognosis and trigger potent antihepatocellular carcinoma (HCC) activity by activating CD39+CD8+ T cells. Gut, 70(10), 1965-1977.
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